Multiple antigenic peptide assay for detection of hiv or siv type retroviruses

ABSTRACT

A method for detecting at least one antibody directed against at least one primate immunodeficiency virus in a biological sample that includes contacting a biological sample with (i) at least one detection multiple antigenic peptide comprising a portion of an immunodominant region of a transmembrane protein of a primate immunodeficiency virus and (ii) at least one differentiation multiple antigenic peptide comprising a portion of a V3-loop of an envelope protein of a primate immunodeficiency virus. Also disclosed is an enzyme immunoassay that includes a first substrate to which are bound at least one of the detection multiple antigenic peptides and a second substrate to which are bound at least one of the differentiation multiple antigenic peptides.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation application of co-pending U.S. application Ser.No. 10/552,182, filed Oct. 5, 2005, which is the U.S. National Stage ofInternational Application No. PCT/US2004/011022, filed Apr. 8, 2004,which was published in English under PCT Article 21(2), and which claimsthe benefit of U.S. Provisional Application No. 60/462,071, filed Apr.11, 2003. The entire disclosures of the prior applications areconsidered to be part of the disclosure of the accompanying applicationand are hereby incorporated by reference.

FIELD

This invention concerns assays for the detection of primateimmunodeficiency viruses.

BACKGROUND

Human immunodeficiency virus (HIV) is subdivided into 2 types, HIV-1 andHIV-2, both of which are believed to be the result of separate zoonotictransmissions on at least eight different occasions (Hahn et al.,Science 287:607-17, 2000; Sharp et al., Philos Trans R Soc Lond B BiolSci 356:867-6, 2001) from chimpanzees and sooty mangabeys, respectively(Huet et al., Nature 345:356-359, 1990; Gao et al., Nature 397:436-441,1999; Hirsch et al., Nature 339:389-392, 1989). While the origin ofHIV-1 from chimpanzees is mainly supported by the phylogeneticclustering of HIV-1 and SIVcpz, substantial evidence supports thezoonotic origin of HIV-2, including similarity in the viral genomeorganization, phylogenetic relatedness, prevalence in the natural host,geographic coincidence and plausible route of transmission (Sharp etal., Philos Trans R Soc Lond B Biol Sci 349:41-47, 1995).

There is no evidence that the other lineages of simian immunodeficiencyvirus (SIV) have crossed into humans. The other lineages include: theSIVagm from four species of African green monkeys; the SIVsyk fromsykes' monkeys; the SIVmnd from a mandrill together with SIVlhoest froml'Hoest monkeys and SIVsun from Sun-tailed monkeys; and the SIVcol froma colobus monkey. SIVs from other non-human primates from Africa havebeen partially sequenced and may represent new lineages. Continued studyof SIV is critical for elucidating the origin and spread of HIV inhumans, and monitoring future viral threats to humans.

A number of studies have provided serological evidence (usingcommercially available HIV tests) of SIV infections in at least 30African non-human primates to date with viral molecular evidence in 24of the infections (Hahn et al., Science 287:607-617, 2000; Lowenstine etal., Int J Cancer 38:563-574, 1986; Nicol et al., J Med Primatol18:227-236, 1989; Peeters et al, Emerg Infect Dis 8:451-457, 2002).Humans are also now being increasingly exposed to the many differentSIVs in different species of wild primates, for example through thehunting and butchering trade in Sub-Saharan Africa, particularly inCameroon. This increasing human exposure to the plethora of SIVsprevalent in different species of wild primates may lead, or has alreadyled, to additional transmissions of SIVs with the potential to cause newepidemics. Unfortunately, new zoonotic transmissions may easily goundetected because of the lack of SIV-specific tests.

There is no commercially available test specifically designed fordetecting all known SIVs. Serological detection of SIVs has so far beendone using HIV tests (Tsujimoto et al., Nature 341:539-541, 1989;Peeters et al., AIDS 6:447-451, 1992; Peeters et al, AIDS Res HumRetroviruses 10:1289-1294, 1994; Georges-Courbot et al., J Virol72:600-608, 1998; Beer et al, J Virol 73:7734-7744, 1999; Hirsch et al.,Virol 73:1036-1045, 1999; Osterhaus et al, Virology 260:116-124, 1999)based on some cross reactivities observed with SIV antibodies to someHIV antigens. It has not been established whether all SIV strains couldbe detected in this way and as such, some can readily be missed (Simonet al., AIDS Res Hum Retroviruses 17:937-952, 2001; Peeters et al.,Emerg Infect Dis 8:451-457, 2002) due to the high genetic diversityamong primate lentiviruses. Indeed, some seronegative monkeys have beenfound to be infected only as determined by PCR and sequencing (Peeterset al., Emerg Infect Dis 8:451-457, 2002). It would therefore be usefulto develop and implement testing methods and strategies sensitive andspecific enough to detect diverse SIV strains in monkeys and humans inthe event of zoonotic jumps to identify primary infection and preventsecondary transmission that could lead to yet another HIV-like epidemic.

SUMMARY OF THE DISCLOSURE

Disclosed herein is a method for detecting a primate immunodeficiencyvirus (PIV) infection by analyzing a biological sample (such as a serumsample) from a test subject to detect the presence of anti-PIVantibodies in the biological sample. The method includes contacting abiological sample with (i) at least one detection multiple antigenicpeptide (MAP) from an immunodominant (“IDR”) region of a transmembraneenvelope protein of a primate immunodeficiency virus and (ii) at leastone differentiation multiple antigenic peptide from a third variableloop (“V3-loop”) of an envelope protein of a primate immunodeficiencyvirus. At least one of the detection (IDR) MAP or the differentiation(V3-loop) MAP can form an immune complex with primate immunodeficiencyvirus-specific antibody present in the biological sample. The resultingimmune complex then is detected, wherein formation of the complex withthe detection MAP indicates infection with a PIV, and formation of thecomplex with the differentiation MAP indicates infection with aparticular type of PIV (such as HIV-1, HIV-2, SIVcpz, SIVsm, etc.).

Also disclosed is an enzyme immunoassay that includes a first substrateto which is bound at least one detection MAP and a second substrate towhich is bound at least one differentiation MAP. The enzyme immunoassaymay be provided in the form of arrays of different detection MAPs anddifferent differentiation MAPs.

Diagnostic kits that include the detection MAP, the differentiation MAP,and instructions for performing an enzyme immunoassay of a biologicalsample using the detection MAP and the differentiation MAP to detect atleast one primate immunodeficiency antibody in the biological sample arealso described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain examples will be described in more detail below with referenceto the following drawings:

FIG. 1 is a graph showing the optical density (OD) results for an enzymeimmunoassay performed on samples from Sykes monkeys infected with SIVsykagainst an array of different SIV MAPs as described herein that utilizedboth a detection component (identified in FIG. 1 as “IDR”) and adifferentiation component (identified in FIG. 1 as “V3”);

FIG. 2 is a graph showing the optical density (OD) results for an enzymeimmunoassay performed on samples from sooty mangabeys and macaquesinfected with SIVsm against an array of different SIV MAPs as describedherein that utilized both a detection component (identified in FIG. 2 as“IDR”) and a differentiation component (identified in FIG. 2 as “V3”);and

FIG. 3 is a graph showing the optical density (OD) results for an enzymeimmunoassay performed on samples from colobus monkeys infected withSIVcol as described herein that utilized both a detection component(identified in FIG. 3 as “IDR”) and a differentiation component(identified in FIG. 3 as “V3”).

DETAILED DESCRIPTION OF SEVERAL EXAMPLES

For ease of understanding, the following terms used herein are describedbelow in more detail:

“Detection component” generally refers to an assay component that candetect the presence of a PIV, especially SIV.

“Diagnostically effective amount” means the amount of detectably labeledspecific binding agent (e.g., a MAP that binds a PIV antibody) that,when utilized, is in sufficient quantity to enable detection of the PIVantibody.

“Differentiation component” generally refers to an assay component thatcan differentiate between types, strains, or sub-strains of PIV,especially SIV.

“Epitope” generally refers to any antigenic determinant on an antigen towhich the paratope of an antibody binds. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics.

“HIV” is the “Human Immunodeficiency Virus”, which is the infectiouspathogen associated with AIDS (Acquired Immunodeficiency Syndrome).“SIV” is the “Simian Immunodeficiency Virus” that is associated with asimilar syndrome in macaque monkeys that have been infected withcross-species transmission. However, simian hosts that harbor naturallyoccurring SIV strains are asymptomatic. HIV-1 and HIV-2 are believed tohave originated in non-human primates, and have been passed to humans byzoonotic transmission. The viruses that initiate infection are usuallytransmitted in the blood, semen or other body fluid of the infectedindividual. The HIV or SIV genome has the basic arrangement of nucleicacid sequences characteristic of all known retroviruses. Long terminalrepeats (LTRs) at each end of the genome regulate viral integration intothe host genome, viral gene expression and viral replication. The gagsequence encodes core structural proteins, and the env sequences encodethe envelope glycoproteins which are required for infection of thecells. The pol sequences encode reverse transcriptase, integrase, andprotease enzymes required for viral replication. The functions of theseand other retroviral genes are described in great detail in Abbas etal., Cellular Immunology, 4^(th) edition, pages 454-467 (2000).

In PIV, Env is a complex that includes a transmembrane gp41 or gp36(e.g., “gp41/gp36”) subunit and an external, noncovalently associatedgp120 subunit. The Env complex is expressed as a trimeric structure ofthree gp120/gp41 pairs that mediates a multistep process of fusion ofthe virion envelope with the membrane of a target CD4 positive T-cell.In some PIVs, the gp41 subunit is truncated to a gp36 subunit.

“Gp41/gp36” denotes the retroviral transmembrane envelope glycoprotein41 or glycoprotein 36 that is found in retroviruses such as PIV. Withrespect to PIVs, gp 41 or gp36 is a highly immunogenic protein whichelicits a strong and sustained antibody response in individuals infectedwith HIV. A significant proportion of the antibody response to gp41/gp36is directed toward a well-characterized immunodominant region (IDR)within gp41/gp 36. The core of the fusion-active gp41 is composed of atrimer of two interacting peptides that contain a 36-residue peptide(N-36) and a 34-residue peptide (C-34), as described more fully in U.S.Pat. No. 6,150,088.

“Gp120” denotes the outer envelope protein found in retroviruses such as

HIV and SIV. The envelope protein is initially synthesized as a longerprecursor protein of 845-870 amino acids in size, designated gp160.Gp160 forms a homotrimer and undergoes glycosylation within the Golgiapparatus. It is then cleaved by a cellular protease into gp120 andgp41. Gp41 contains a transmembrane domain and remains in a trimericconfiguration; it interacts with gp120 in a non-covalent manner. Gp120contains most of the external, surface-exposed, domains of the envelopeglycoprotein complex, and it is gp 120 which binds both to the cellularCD4 receptor and to the cellular chemokine receptors (e.g., CCRS).

The gp120 core has a unique molecular structure that includes twodomains: an “inner” domain (which faces gp41) and an “outer” domain(which is mostly exposed on the surface of the oligomeric envelopeglycoprotein complex). The two gp120 domains are separated by a“bridging sheet” that is not part of either domain. Binding to CD4causes a conformational change in gp120 which exposes the bridging sheetand may move the inner and outer domains relative to each other. TheCD4-binding pocket within gp120 comprises a number of residues whichinteract directly with Phe43 of CD4. The most important of these areGlu370, Trp427 and Asp368 (the latter residue also forms a salt bridgewith Arg59 of CD4). These three residues are conserved in all primatelentiviruses.

One region which has been shown to elicit neutralizing antibodies is thehypervariable V3 loop or region (V3) of the gp120; this is an epitopethat elicits potent neutralizing Abs in man and experimental animals(summarized in Javaherian et al., Science 250:1590-93, 1990). The highlyvariable V3 loop of the surface protein distinguishes among PIVlineages. The V3 loop may be defined by two cysteine residues that forma disulfide bond forming the loop structure. The full length V3 sequencetypically contains 33-35 amino acid residues. Examples of at leastpartial sequences or sub-sequences for the V3 of gp120 areNNTRGEVQIGPGMTFYNIENVVGDTRSA (SIVcpzGab) (SEQ ID NO: 23),NRSVVSTPSATGLLFYHGLEPGKNLKKG (SIVmnd) (SEQ ID NO: 24),NKTVLPVTIMAGLVFHSQKYNTRLRRQA (SIVagm) (SEQ ID NO: 25), andNKTVLPVTIMSGLVFHSQPINERPKQA (SIVsm) (SEQ ID NO: 26) (Simon et al., AIDSRes Hum Retroviruses 17:937-952, 2001). Further examples of V3 sequencescan be found in Pau et al., AIDS Research and Human Retroviruses,10:1369-1377, 1994).

“Immunodominant region” or “IDR” refers to the subunit of an antigenicdeterminant that elicits a strong immunological response. It has beenestablished by use of chemical synthetic peptides that the IDR epitopesof HIV-1 or HIV-2 envelope proteins are located in the relativelyconserved regions. An example of the location of the IDR for certainstrains of HIV-1 gp41 is at amino acid positions 584-618 (Shin et al.,Biochemistry and Molecular Biology International 43:4:713-721, 1997),and, more particularly, at 598-609 (Gnann et al., Journal of Virology61:2639-2641, 1987). An example of a location of the IDR for HIV-2 gp36is at amino acid positions 574-602 (Shin et al., Biochemistry andMolecular Biology International 43:4:713-721, 1997). The IDRs of thevarious SIVs are at the same or similar positions. The full lengthsequence of the IDR of gp41/gp 36 typically contains 34-44 amino acidresidues. Examples of at least partial sequences or sub-sequences forthe IDR of gp41/gp36 are LAVERYLQDQQILGLWGCSGKAVC (SIVcpzGab) (SEQ IDNO: 27), TSLENYIKDQALLSQWGCSWAQVC (SIVmnd) (SEQ ID NO: 28),TALEKYLEDQARLNIWGCAFRQVC (SIVagm) (SEQ ID NO: 29), andTAIEKYLKDQAKLNSWGCAFRQVC (SIVsm) (SEQ ID NO: 30) (Simon et al., AIDS ResHum Retroviruses 17:937-952, 2001).

“Multiple antigen peptide” refers to a combination antigen/antigencarrier that is composed of two or more, identical or different,antigenic molecules. In some instances, the antigenic molecules arecovalently attached to a dendritic core that is composed of bifunctionalunits, such as lysine molecules. For example, a lysine is attached viapeptide bonds through each of its amino groups to two additionallysines. This second generation molecule has four free amino groups eachof which can be covalently linked to an additional lysine to form athird generation molecule with eight free amino groups. A peptide may beattached to each of these free groups to form an octavalent multiplepeptide antigen. Alternatively, the second generation molecule havingfour free amino groups can be used to form a tetravalent MAP. In otherexamples, aspartic acid or glutamic acid can be used to form thedendritic core of a multiple peptide antigen. The dendritic core, andthe entire MAP, may be conveniently synthesized on a solid resin usingthe classic Merrifield synthesis procedure.

“Peptide” refers to any molecule or moiety comprising two or more aminoacid residues joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Peptides may contain amino acids otherthan the 20 gene-encoded amino acids. Peptides include amino acidsequences modified either by natural processes, such aspost-translational processing, or by chemical modification techniques.Glycosylated and non-glycosylated forms of peptides are also embraced by“peptides.” Peptides can include D or L-amino acid residues. “Peptides”also includes peptide analogues such as synthetic constructs using thecarbon skeleton of peptides but omitting the -CONH- peptide bonds.

“PIV” is a primate immunodeficiency virus, which includes both HIV andSIV, for example the human viruses HIV-1 and HIV-2; the chimpanzee virusSIVcpz such as, for example, SIVcpzGab, SIVcpzCam, SIVcpzAnt, andSIVcpzUS; the sooty mangabey virus SIVsm; the African green monkey virusSIVagm such as, for example, SIVagm-1 and SIVagm-2; the mandrill virusSIVmnd such as, for example, SIVmnd14 and SIV mndGB 1, as well as a hostof others including SIVsun/lhoest, SIVcol, SIVrcm, SIVsyk, SIVdeb,SIVgsn, SIVmon, SIVmus, and SIVtal. PIV is inclusive of all strains(e.g., SIVcpz) and sub-strains (e.g., SIVcpzGab).

“PIV antibody detection specificity” refers to the capability of anassay to detect true PIV-negative biological samples (i.e., accuratelydifferentiate between PIV-positive and PIV-negative biological samples).For example, specificity is expressed herein in the terms oftrue-negative test results divided by all subjects not infected with thevirus. In other words, specificity=(d/(b+d)) wherein d=true negativesand b=false positives. An increase in the specificity of an assayresults in fewer false positives.

“PIV antibody detection sensitivity” refers to the capability of anassay to detect true PIV-positive biological samples. For example,sensitivity is expressed herein in the terms of true-positive testresults divided by all subjects infected with the virus. In other words,sensitivity=(a/(a+c)) wherein a=true positives and c=false negatives. Anincrease in the sensitivity of an assay results in fewer falsenegatives.

“Sequence identity” refers to the similarity between two nucleic acidsequences, or two amino acid sequences, and is expressed in terms of thesimilarity between the sequences, otherwise referred to as sequenceidentity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are. Homologs ororthologs of a peptide sequence (such as the sequence of an IDR ofgp41/36 or the V3 peptide), and the corresponding DNA sequence, willpossess a relatively high degree of sequence identity when aligned usingstandard methods. This homology will be more significant when theorthologous proteins or DNAs are derived from species which are moreclosely related (e.g., human and chimpanzee sequences), compared tospecies more distantly related (e.g., human and murine sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-244, 1988); Higgins &Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nuc. Acids Res. 16,10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8,155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24, 307-31, 1994.Altschul et al., J. Mol. Biol. 215:403-410, 1990, presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence identity using this program. BLAST searchingpermits determination of sequence identity between a given sequence, forexample, a nucleotide sequence, and a reference sequence. Nucleotidesequences with increasing similarity to a reference sequence showincreasing percentage identities, for example at least 50%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least98%.

Homologs of the disclosed peptides typically possess at least 60%sequence identity counted over full-length alignment with the referencesequence using the NCBI Blast 2.0, gapped blastp set to defaultparameters. For comparisons of amino acid sequences of greater thanabout 30 amino acids, the Blast 2 sequences function is employed usingthe default BLOSUM62 matrix set to default parameters, (gap existencecost of 11, and a per residue gap cost of 1). When aligning shortpeptides (fewer than around 30 amino acids), the alignment should beperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequence will show increasing percentage identities when assessed bythis method, such as at least 70%, at least 75%, at least 80%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs will typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and can possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are described in the NCBI website. These sequenceidentity ranges are provided for guidance only; it is entirely possiblethat strongly significant homologs could be obtained that fall outsideof the ranges provided.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (Tm) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, CSHL,New York and Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, N.Y. Nucleic acid molecules that hybridize under stringentconditions to a given sequence will typically hybridize under washconditions of 2×SSC at 50° C.

Nucleic acid sequences that do not show a high degree of identity cannevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

The above term descriptions are provided solely to aid the reader, andshould not be construed to have a scope less than that understood by aperson of ordinary skill in the art or as limiting the scope of theappended claims.

The immunoassay disclosed herein is useful for detecting primateimmunodeficiency viruses (PIV) in a biological sample. The immunoassayis especially useful for detecting SIV in non-human primates andSIV-like infections in humans. The assay also is useful for detectingpreviously unrecognized SIV-like retroviruses in humans and non-humanprimates. Furthermore, the assay can be used for differentiating betweenspecific SIV strains.

The assay method generally involves contacting the biological samplewith multiple antigenic peptides (MAPs) from an immunoreactive region ofPIV envelope proteins. The biological sample is contacted with the MAPsunder conditions which are suitable for forming immune complexes betweenPIV-specific antibodies in the biological sample and the MAPs. The assaycan detect a wide variety of PIVs by providing an array of MAPs frommultiple PIVs, wherein the MAPs for each PIV include a detectioncomponent and a differentiation component. Of course, certain examplesof the assay may include only the detection component or differentiationcomponent for a given specific PIV. In a particular example, thedetection component is an IDR peptide of a PIV transmembrane protein(for example, from gp41 or gp 36), and the differentiation component isa V3 peptide of an envelope protein (for example, gp120). The IDRcomponent provides a high degree of sensitivity to antibodies againstmany PIV strains, while the V3 component is a less sensitive, but morespecific variable component that differentiates between PIV species.Certain examples of the IDR peptide and the V3 peptide have sequencelengths that are set forth below in order to enhance their specificityfor PIV, particularly SIV. In addition, the MAP construct provides anassay with a significantly improved degree of analytical sensitivity asdetailed below.

The presently described methods are particularly useful for serologicaltesting employing serum or plasma samples, although the biologicalsamples could alternatively be in the form of any body fluid such aswhole blood, urine, saliva and other fluids or body tissue. Thebiological samples can be taken from humans, non-human primates, andexperimental animal models such as mice or rabbits.

MAPs, which are described in more detail below, typically includepeptide antigen sequences covalently linked to a branching, dendriticcore residue or matrix composed of bifunctional units (e.g., lysines).More specifically, a biological sample is contacted with at least onefirst MAP that serves as a detection component (for example, a MAP froman immunodominant region of a transmembrane protein of a PIV) and atleast one second MAP that serves as a differentiation component (forexample, a MAP from a V3-loop of an envelope protein of a PIV). Thefirst MAPs in these examples are referred to herein as “IDR MAPs” andthe second MAPs are referred to herein as “V3 MAPS.”

The assaying of a sample from an individual subject with the IDR and V3MAPs may be performed serially or simultaneously. If performed serially,the assaying can be conducted in any order. In other words, a firstportion of a biological sample from an individual subject may becontacted with the IDR MAPs. A second portion of the same biologicalsample may be contacted with the V3 MAPs, either simultaneously, priorto, or after, the IDR assaying. In certain variations, IDR and V3testing could be performed simultaneously by providing IDR and V3 MAPson the same panel or microplate.

The MAP assays also can be used to quantify anti-PIV antibodies in sera,measure the affinity of the antigen-antibody complex, or the avidity ofthe antigen-antibody complex. Such measurements can be obtained by usingtechniques known in the art.

Specific examples of MAPs that are useful in the presently disclosedassay are composed of two or more, usually identical, antigenic peptidescovalently attached to a dendritic core matrix which is composed ofbifunctional units. Suitable core molecules for constructing MAPsinclude ammonia, ethylenediamine, aspartic acid, glutamic acid andlysine. For example, a lysine core molecule is attached via peptidebonds through each of its amino groups to two additional lysines. Thissecond generation molecule has four free amino groups, each of which canbe covalently linked to an additional lysine to form a third generationmolecule with eight free amino groups. A peptide may be attached via itsC-end to each of these free groups to form an octavalent multipleantigenic peptide (also referred to as a “MAP8” structure).Alternatively, the second generation molecule having four free aminogroups can be used to form a tetravalent or tetrameric MAP, i.e., a MAPhaving four peptides covalently linked to the core (also referred to asa “MAP4” structure). The carboxyl group of the first lysine residue maybe left free, amidated, or coupled to β-alanine or another blockingcompound. As used herein, the “linear portion or molecule” of a MAPsystem structure refers to the antigenic peptides that are linked to thecore matrix. Thus, a cluster of antigenic epitopes form the surface of aMAP and a small matrix forms its core.

The dendritic core, and the entire MAP may be conveniently synthesizedon a solid resin using a classic Merrifield synthesis procedure. MAPsynthesis, is generally described, for example, in U.S. Pat. Nos.5,580,563, and 6,379,679, and Tam, Proc. Natl. Acad. Sci. USA85:5409-5413, 1988.

Examples of a schematic structure of a tetrameric or MAP4 peptide arerepresented below in Formula 1

wherein R′ represents an amino acid sequence, especially a linearsequence. Each R′ may be the same or different. The number of amino acidresidues in R′ may vary, and is described in more detail below.

A comprehensive array of peptides covering gp41/36 and gp120 proteinsregions of all recognized (i.e. characterized) SIV strains can be used.The assay is an open-ended assay in that new MAPs can be added to theassay template as additional SIV strains are identified andcharacterized. Consequently, the assay has the flexibility to becontemporaneously updated so that it may be capable of screening for allSIV strains recognized or known at the time of assaying. Of course,variants of the assay can be made with less than all of the known SIVstrains. According to one example, the assay does not include anypeptides from HIV (in other words, the assay only includes peptides fromSIV).

The specific MAPs may be arranged in any suitable configuration in thearray. For example, the MAPs may be bound onto an 8×12 microwell platein which a first MAP (e.g., a MAP from SIVcpzGab) is coated into all thewells in the first column, a second MAP (e.g., a MAP from SIVsm), iscoated into all the wells in the second column and so forth.

Antibodies generally cannot bind to the whole antigen molecule.Generally, a specific antibody binds specifically to one individualepitope on a protein antigen. In the presently disclosed assays, theantigenic reactive substance is from certain immunodominant epitopes.More specifically, the antigenic peptide R′ sequences in the MAPs areselected from immunodominant regions in the respective nativetransmembrane or envelope proteins. As mentioned above, the R′ antigenpeptides may be the same or different in individual MAP molecules.Employing different R′ antigen peptides in individual MAP molecules canallow for the inclusion of a wider variety of PIVs in a single assay.

Peptides from all primate lentiviral lineages for which envelopesequences were available at the time of preparing the provisional patentapplication from which the present application claims priority were usedin one example of the assay. The peptides were those that can beexpressed from 2 separate regions of the envelope protein of the viralgenome. The first region was the immunodominant region (IDR) of thetransmembrane protein (gp41/gp36), and the second region was the V3-loopof the envelope protein (gp120). As described above, the IDR epitope wasselected for its high sensitivity and the V3-loop was selected for itshigh specificity. The detection component and/or the differentiationcomponent also could be from other regions of the envelope, gag, or polprotein.

Given that longer peptides may give rise to non-specific reactivitiesoutside or within primate lentiviruses, especially useful peptidesequences for MAP synthesis and assaying have less than about 16 aminoacid residues per linear portion of each MAP. In illustrative examples,each linear portion of each IDR MAP may include about 5 to about 15,more particularly about 7 to about 11, amino acid residues, and eachlinear portion of each V3 MAP may include about 5 to about 15, moreparticularly about 7 to about 15, amino acid residues. Peptide sequencesof such lengths are particularly useful for constructing MAPs from SIVstrains. It should be recognized that the number of amino acid residuesprovided above does not include any non-viral coded amino acid residuesthat may be used as spacers in the R′ linear portion of a MAP such asthat depicted above in Formula 1.

The R′ sequences for the IDR MAP may be selected, for example, from theIDR sequences discussed above such as SEQ ID NOS: 27-30. Especiallyuseful R′ sequences for the IDR MAP are those that contain a minimum of7 amino acid residues defined by two cysteine residues. According to oneexample of the detection MAP, the R′ sequence of the IDR MAP constructmay have (or include) a consensus sequence represented by

X₁GCX₄X₅X₆X₇X₈CX₁₀T

wherein X₁ is W, I or F;

-   -   X₄ is S, A or Q;    -   X₅ is G, D, F, W or N;    -   X₆ is K, R, M, S, or A;    -   X₇ is A, V or Q;    -   X₈ is V, or I; and    -   X₁₀ is Y, H or R.

Alternatively, the R′ sequence of the detection MAP is SEQ ID NOS: 1, 8,9, or a sequence having at least 80%, 90% or 95% sequence identity toone or more of those sequences.

The R′ sequences for the V3 MAP may be selected, for example, from theV3 sequences discussed above such as SEQ ID NOS: 23-26. In an example ofthe differentiation MAP, R′ is one or more of SEQ ID NOS: 14-22, or asequence having at least 80%, 90% or 95% sequence identity to one moreof those sequences.

One illustrative MAP array used in an example of the presently describedassay is shown below in Table 1.

TABLE 1 Composition of IDR and V3 peptides used in a MAP assay. SEQ SEQIDR gp41/36 ID ID Virus peptides NO: Virus V3 peptides NO: SIVcpzGabWGCSGKAVCYT 1 SIVcpzGab RGEVQIGPGMTFYNI 14 SIVcpzCam WGCSGKAICYT 2 SIVsmVLPVTIMSGLVFHSQ 15 SIVcpzAnt WGCADKVICHT 3 SIVagm LPVTIMAGLVFHSQ 16SIVsm WGCAFRQVCHT 4 SIVsyk IKNIQLAAGYFLPVI 17 SIVagm-1 WGCAWKQVCHT 5SIVlhoest EVSTISSTGLLFYYG 18 SIVagm-2 WGCAFKQVCHT 6 SIVcolHRNLNTANGAKFYYE 19 SIVsun/lhoest WGCQWKQVCHT 7 SIVrcm VKGISLATGVFISLR 20SIVcol IGCANMQICRT 8 SIVmnd14 IVSVPSASGLIFYHG 21 SIVrcm FGCAWRQVCHT 9SIVdeb YRAVHMATGLSFYTT 22 SIVmnd14 WGCSFSQVCHT 10 SIVmndGB1 WGCSWAQVCHT11 SIVsyk WGCAFKQICHT 12 SIVdeb WGCAFKQICHT 13

An array of the specific MAPs shown in Table 1 is immobilized on amicrotiter plate as described in more detail below. The sequences listedrepresent specific examples of the R′ group of Formula 1. In the case ofan IDR MAP, each R′ also includes β-alanine (βA) and d-aspartic acid(dD) between the specific epitope sequence listed in Table 1 and eachlysine matrix molecule (e.g., SEQ ID NO: 1-βA-dD-K-). The β-alanine (βA)residue serves as a spacer amino acid between the reactive epitopesequence and the lysine core. The d-aspartic acid (dD) residue serves asa spacer amino acid and improves the solubility of the MAP. Lysine andglutamic acid also can be used as spacer amino acids. In the case of aV3 MAP, each R′ also includes diaminopropionic acid (X) as a spaceramino acid between the specific epitope sequence listed in Table 1 andeach lysine matrix molecule (i.e., SEQ ID NO: 14-X-K-). As an example,Formulae 2 and 3 show specific MAP structures with SEQ ID NO: 1 and SEQID NO: 14, respectively.

The specificity of peptides generally tends to increase as the length ofthe peptides decreases, but shorter peptides may also have reducedreactivity, which can reduce the sensitivity of the test. The MAPstructure can compensate for this reduced sensitivity. In particular,the plurality of shorter linear peptides in the presently disclosed MAPsenables optimization for specificity and sensitivity. The specificity isenhanced by shorter linear peptide portions that are more antigenicityfocused. The sensitivity is enhanced by the plurality of shorter linearpeptides. For instance, the analytical discernability of the assayresults is increased (e.g., the optical density readout exhibits a moreintense color). Although not bound by any theory, it is believed thatsince only a portion of the MAP molecule is in contact with the solidphase substrate, the other portions of the MAP molecule are free forantibody binding. In addition, MAPs provide increased antigen density,and thus an increased number of antibody binding sites per unit surfacearea.

The PIV antibody detection specificity of the presently disclosed assaysmay be, for example, at least about 95%, and more particularlysubstantially 100%. The PIV antibody detection sensitivity of thepresently disclosed assays may be, for example, at least about 95%, andmore particularly substantially 100%. When IDR and V3 components areused in combination, 100% specificity and sensitivity may be achieved.

The MAPs may be contacted with a biological sample by any suitable assaytechnique. For instance, the MAPs may be bound to a solid phasesubstrate that is then contacted with the biological sample. The solidphase substrate could be a microtiter plate in which each specific MAPis bound to an individual microwell. Alternatively, the MAPs could bebound to solid beads, magnetic beads or similar type of particulatemedia, membranes, discs, gels, flat sheets, test strips, fibers andother configurations and types of materials that permit antigens to beattached to the support. Attachment may be made by non-covalent orcovalent means. Preferably, attachment will be made by adsorption of theantigen to a well in a microtiter plate, a membrane such asnitrocellulose, or latex beads. Other possible attachment techniquesinclude biotinylation of the MAPs and capture of the MAPs with avidin ona solid phase. A diagnostically effective amount of MAP is coated ontothe solid phase substrate. Such an amount may range, for example, fromabout 0.1 to about 1 μg. A single MAP, or a mixture of different MAPs,may be coated in each well.

One optional approach contemplates providing a first solid phasesubstrate that includes an array of desired IDR MAPs, and a second solidphase substrate that includes an array of desired V3 MAPs. Serumsample(s) from an individual subject then are contacted to both the IDRMAP substrate and the V3 MAP substrate. In another optional approach,IDR MAPs and V3 MAPs are arrayed on a single solid phase substrate and aserum sample is flowed over, around, and/or through the substrate suchthat the sample contacts both the IDR MAPs and the V3 MAPs. According toa further optional approach IDR MAPs and V3 MAPs can be mixed together,and the resulting mixture is coated on individual wells.

Any type of immunologic method or methods may be employed to detect theformation of an immune complex between the IDR MAP and/or V3 MAP andPIV-specific antibody present in the biological sample. Illustrativetechniques include radioimmunoassays, competitive immunoassays, enzymeimmunoassays (EIA) such as enzyme-linked immunosorbent assays (ELISA),immunoflourescence, and lateral flow immunoassays. In general, once thebiological sample is exposed to the immobilized MAPs for a sufficienttime (e.g., about 10 minutes to about 24 hours), the support is washedto eliminate any material from the biological sample that is not boundto the MAPs. Such washing step(s) may be performed with saline and otheradditives typically included in a washing solution. A labeled reagentthen is added to the material on the solid support to detect the bindingbetween the MAPs on the solid support and PIV-specific antibody in thebiological sample. Such a reagent may be an anti-human immunoglobulin,such as goat anti-human immunoglobulin, protein A or protein G or anyanti-animal immunoglobulin. In one example, the label can be acolorimetric indicator which upon contact with a substrate produces adetectable color signal. The presence and/or intensity of the colorprovides evidence of the presence of the antibody. Other labels orreporter groups include a radioisotope, a fluorescent compound, afluorescence emitting metal of the lanthanide series, a chemiluminescentor phosphorescent molecule, a paramagnetic group, or an enzyme.

The methods disclosed herein are particularly useful with ELISA. Ingeneral, ELISA involves immobilizing an IDR MAP or V3 MAP onto a solidphase substrate (e.g., a microwell or microtiter plate). Sera or plasmaare added to the microwells, and then the microwells are washed toremove unbound antibody. A second antibody, which is an anti-humanimmunoglobulin (or any anti-animal immunoglobulin) antibody linked withan enzyme, is then added to the wells. Then the substrate for the enzymeis added to the washed well and the amount of enzymatically alteredsubstrate is measured. The enzyme and substrate are chosen so thatenzymatic modification of the substrate produces a change in color ofthe substrate solution. The amount of changed substrate (which may bemeasured with a spectrophotometer) as a result of theenzyme-antibody-antigen reaction is proportional to the amount ofantibody bound to the immobilized MAPs (i.e., the formation ofantigen-antibody complex). Illustrative enzymes include alkalinephosphatase, glucose oxidase, beta-galactosidase, catalase, malatedehydrogenase, horseradish peroxidase, yeast alcohol dehydrogenase, andsimilar enzymes. The assay can be performed using automated ELISAprocessing equipment such as those instruments commercially availablefrom Grifols-Quest, Inc. or Axsyn.

By recording or tracking the coordinates of each specific MAPimmobilized on the microplate, which PIV-specific MAP(s) that haveformed immune complexes can be determined. Thus, the assay disclosedherein can also differentiate between specific PIV strains.

As mentioned above, the assays disclosed herein can also be utilized forscreening for divergent SIV strains. More specifically, if a certainbiological sample is positive for both the IDR and the V3 components,then the molecular structure of the sample can be further characterizedby techniques such as polymerase chain reaction (PCR) and genesequencing. The characterized molecular structure then can be comparedto reference SIV strains to determine if the sample of interest is adivergent or previously unrecognized strain.

Also disclosed herein are diagnostic kits that can be used to performthe above-described detection methods. A kit could include microplateson which the desired MAPs have already been immobilized. Alternatively,the kit could include containers containing MAPs in solution for coatingonto microplates by the user. Any number of different MAPs could beincluded, but the kit preferably would include MAPs corresponding to allthe SIV recognized and characterized at the time the kit was prepared.

Also provided in the kit are containers containing labeled reagents orconjugates which detect the binding of antibody to the immobilized MAP,such as goat anti-human immunoglobulin or the like. The label on thereagent may be selected from any type of diagnostic labels, such asradioactive compounds, fluorescent compounds and proteins, colorimetricenzymes, etc. The kit also may contain miscellaneous reagents andapparatus for reading labels (e.g., certain substrates that interactwith an enzymatic label to produce a color signal), apparatus for takingblood samples, as well as other appropriate vials and other diagnosticassay components. One specific example of a kit could include variousMAPs, a bicarbonate buffer for dissolving the MAPs, a goat anti-humanimmunoglobulin conjugate, a milk buffer, phosphate-buffered saline, andan enzyme substrate such as tetramethyl-benzylidene/hydrogen peroxide.Instructions for performing the assay may also be included with the kit.

The kit also could include a reference or key member having colorimetricstandards corresponding to the optical densities indicating PIV-positivehits. Such a reference or key member could be used for visuallydetecting PIV-positive hits with the unaided eye in the event thatspectrophotometry instruments are unavailable in the field. Thereference member, for example, could be a paper or plastic substrate ora container having different colored regions on its surface. Eachcomponent of the kit could be packaged together or separately.

The specific examples described below are for illustrative purposes andshould not be considered as limiting the scope of the appended claims.

MAP Synthesis

All the peptide sequences used in the exemplified assay are shown abovein Table 1. The MAPs were synthesized using a solid-phase method,9-fluorenyl-methoxycarbonyl (Fmoc) chemistry according to themanufacturer's protocol on an automatic synthesizer (model 432A, AppliedBiosystems, Inc., Foster City, Calif.). The synthesis started from theright side of the illustrative structure shown above in Formula 1, andthen proceeded to the left side through the R′ sequence. Aftersynthesis, the MAPs were partially purified by reverse phasehigh-performance liquid chromatography (BioRad, Richmond, Calif.),lyophilized, and stored desiccated at room temperature until use.

MAP Enzyme Immunoassay

The MAPs (10 μg/μl) were first dissolved in dimethyl sulfoxide (AldrichMilwaukee, Wis., USA), then diluted in cold bicarbonate buffer (0.1 M,pH 9.4) to a final concentration of 0.25 μg/100 μl. The MAP solutionsthen were coated (110 μl/well) onto microtiter plates (Maxisorb, NalgeNunc International, Rochester, N.Y.) at 4° C. overnight. MAPs that werederived from the same primate species or which have very close identityamino acid sequences were mixed to a maximum of two per well and thencoated. In particular, the SIVcpzGab and SIVcpzCam IDR MAPs were mixedtogether, the SIVagm-1 and SIVagm-2 IDR MAPs were mixed together, andthe SIVsyk and SIVdeb IDR MAP is the same MAP. Unbound MAP was removedby washing 2 times with phosphate buffered saline (PBS, pH 7.5)containing 0.05% TX-100 (Sigma Chemical Co., St. Louis, Mo.) (PBS-TX),dried at 37° C., sealed in bags with dessicant, and stored at -20° C.until use. Non-specific binding sites were blocked with 5% nonfat drymilk in PBS containing 0.1% Triton X-100 (milk buffer) (octylphenolethylene oxide condensate) for 30 minutes at 37° C. just prior toperforming the assay. Plasma samples were diluted 1:500 in milk buffer,and 100 μl was added to the MAP-coated well and incubated for 1 hour at37° C. Bound antibodies were detected with 1:8000 dilution (30 minutesincubation at 37° C.) of goat anti-human IgG(H+L)-peroxidase conjugate(BioRad, Hercules, Calif.) and tetramethyl-benzydine/hydrogen peroxidasesubstrates (BioFX, Owings Mills, Md.) for 10 minutes at roomtemperature. Color development was stopped with 1M sulfuric acid andoptical density (OD) was measured at 450 against a reference of 630 nm.

Reference Panels

The assay was evaluated using known and well characterized simian andhuman samples obtained from divergent representatives of primate speciesknown to carry lentiviruses belonging to 6 viral lineages.

1. Non-Human Primate Reference Panel

This panel included 54 sera from various monkeys experimentally ornaturally infected with SIVs, and include all SIV strains for whichenvelope V3 and IDR sequences were available. In general, viral DNA froma number of these samples was amplified while others were onlyserologically confirmed by EIA and western blot. Table 1 below shows theSIV strains included in the panel: 6 samples from sooty mangabeys, 10from macaques (4 rhesus and 6 stumptail) accidentally infected withSIVsm, 9 from African green monkeys infected with SIVagm; 4 specimensfrom sykes monkeys infected with SIVsyk; 3 specimens from chimpanzeesinfected with SIVcpz (SIVcpzGab, SIVcpzAnt and SIVcpzUS); 2 from macaquemonkeys infected with SIV1′Hoest and SIVsun; 4 from colobus monkeysinfected with SIVcol; 8 from mandrills, 1 from a drill infected withSIVmnd and SIVdrl, 2 from red capped mangabeys infected with SIVrcm; 4from talapoin monkeys infected with SIVtal, and one specimen from a DeBrazza monkey likely infected with SIVdeb, based on serological results.Since the assay is intended to test humans for SIV-like infections, twospecimens from humans occupationally infected with SIVsm were alsoincluded in the panel.

TABLE 2 Non human primate reference panel Species Common name Virus SIV+SIV− Cercocebus atys Sooty mangabey SIVsm 6 6 C. torquatus Red-cappedSIVrcm 2 15 mangabey Macaca spp Macaque SIVsm 10 8 Chlorocebus. Vervetmonkey SIVagmVer 4 3 pygerythrus C. tantalus Tantalus monkey SIVagmTan 44 C sabaeus Green monkey SIVagmSab — 3 Cercopithecus Sykes monkey SIVsyk4 4 albogularis C. l'hoesti L'Hoest monkey SIVlhoest 2 1 C. neglectus DeBrazza monkey SIVdeb 1 1 Colobus. guereza Guereza colobus SIVcol 4 32Mandrillus. sphinx Mandrill SIVmnd 8 5 M. leucophaeus Drill SIVdrl 1 3Myopithecus. talapoin Talapoin monkey SIVtal 4 5 Pan troglodytes Cent.Africa SIVcpz 2 3 troglodytes chimpanzee (p.t.t.) P. t. schweinfurthiiEast Africa SIVcpz 1 — chimpanzee (p.t.s.) Total 54 93

To determine the specificity of the assay, 93 SIV negative sera fromvarious monkey species (6 sooty mangabeys, 10 African green monkeys, 3chimps, 4 Sykes, 1 1′Hoest, 5 mandrills, 3 drills, 8 macaques, 32colobus, 5 talapoins, 15 red-capped mangabeys, and one De Brazza) weretested. Seronegative status of these monkeys was established based onthe absence of reactivity to commercial ELISA immunoassays. Seventeen ofthese monkeys were infected with SFV, STLV.

2. Human Reference Panel

To assess the specificity of the assay for application in human testing,198 HIV seronegative samples collected in US blood banks were tested.Specimens from the US were preferred as reference over seronegativesamples from Cameroon or other parts of Africa because the close contactof humans and monkeys in this region makes African specimensinappropriate for the purpose.

To determine the reactivity of HIV-1/2 antibodies with the SIV MAPs,included in the evaluation were specimens from individuals infected withHIV. These specimens were as follows: 70 non-B HIV-1 group M fromAfrica, including subtypes A (n=27), C (n=1), D (n=14), AE (n=1), F(n=7), G (n=18), H (n=1) and J (n=1) in addition to 50 subtype B fromthe US; 4 HIV-1 group 0 from Cameroon; 44 HIV-2 from Nigeria and IvoryCoast; and 5 HIV-1+2 from Nigeria.

Other common African infections were also checked that might cross-reactwith SIV MAPs by employing specimens from individuals infected withother endemic infectious agents to evaluate any cross-reactivity withthe SIV MAPs. These included 18 specimens from malaria patients infectedwith the 4 main species of Plasmodium: P. falciparum (n=4), P. malariae(n=2), P.ovale (n=5), and P.vivax (n=7); 44 specimens from HTLV positiveindividuals; 3 specimens from individuals infected with measles; and 3specimens from individuals infected with simian foamy virus (SFV).

The MAP assay described in Table 1 was also employed to determinewhether humans are becoming infected with SIV through exposure toinfected nonhuman primate blood and body fluids. Three groups of HIVnegative plasma samples from Cameroon were available for comparisons:from persons with a high level of exposure (HE) throughhunting/butchering in remote villages (n=76), persons with a lower levelof exposure (LE) in remote villages (n=77), and a general (G) population(n=1071).

Results

The results obtained from various panels of samples were analyzed usingthe box-plot technique. The test performance was evaluated bycalculating the sensitivity and specificity.

1. Non-Human Reference Panel

Three specimens originated from chimpanzees (two P. trogloytesschweinfurthii and one P. t.troglodytes) infected with SIVcpz. All threereacted with the N and SIVcpz IDR MAPs, with one showing additionalreactivity within the lineage with group M peptide. There was noreactivity with other peptides outside this lineage. One of the 2specimens from P t. s. did not react on the V3 MAP component, whichmight be due to low antibody titer since the IDR reactivity wasrelatively lower.

For the second lineage (HIV-2/SIVsm), specimens from 6 sooty mangabeysand 10 macaques (Rhesus and stumptail) infected with SIVsm were used.All 16 specimens were detected in both IDR and V3 components. Althoughsome cross-reactivity was observed in the IDR region, the V3 componentshowed specific reactivity only to the second lineage. The results aredepicted in FIG. 2 which clearly shows the IDR cross-reactivitysensitivity and the improved specificity obtained with the V3 component.Hence the IDR identifies the presence of the PIV, and the V3 componentdifferentiates the particular virus that is present.

The third group of simian samples consisted of 9 African green monkeysinfected with SIVagm. All the specimens were determined to be positiveby the assay. There were limited and low reactivity with IDR MAPs fromother lineages (HIV-2/SIVsm, SIVsyk) but the highest reactivity was tothe homologous MAPs. The V3 MAP was less cross-reactive and thereforemore specific to the SIVagm lineage.

Five sera from infected sykes monkeys were also determined to bepositive by the assay. There was some cross reactivity observed in IDRmainly with the SIVagm MAPs, as it was reversibly the case with SIVagmspecimens reacting against sykes MAPs. The V3 reactivity was veryspecific only to sykes MAPs. The results are depicted in FIG. 1 whichclearly shows the IDR cross-reactivity sensitivity and the V3specificity.

There were only 2 SIV positive sera from l′Hoest monkeys and both weredetected. Cross-reactivity was observed mainly with the HIV-2/SIVsm andAGM gp36 peptides. This is probably due to their similarity in this generegion. The V3 again was specific although one of the specimens showedcross-reactivity with HIV-1 group 0 peptide. Specimens from 8 mandrillsand 1 drill infected with SIVmnd and SIVdrl were tested and all reactedwith the homologous peptides with some cross-reactivity with HIV-2, AGM,RCM, Sykes and l′Hoest in the IDR while cross-reactivity in the V3component was only directed to the l'Hoest peptides.

The reactivity of sera from SIVcol infected colobus monkeys was the bestin terms of sensitivity and specificity for both IDR and V3 components.All 4 positive specimens reacted solely with SIVcol peptides in bothassays as depicted in FIG. 3. This may not be surprising given the highgenetic distance of SIVcol from all others lineages. Two SIV positivespecimens from red-capped mangabeys infected with SIVrcm were alsoevaluated, and all were detected by the presently described system inboth IDR and V3.

Although the talapoin monkey peptides were not included in the assay(because sequences were not available), 4 specimens were evaluated frominfected talapoin monkeys. All were detected in the IDR component of thetest by their cross-reactivity to HIV2, AGM, RCM and Sykes MAPs. Therewas no reactivity with any of the V3 MAPs included in our assay.However, the cross-reactivity in the IDR component demonstrates thatpreviously un-identified SIV strains could be detected by the presentlydescribed assays.

The 93 SIV negative specimens from various monkeys did not show anyreactivity to their homologous MAPs. The specimens from SFV-infected,but SIV negative, monkeys did not show any reactivity with the 2 generegions of the assay.

Samples from two SIVmnd-infected mandrills and two SIVdeb-infectedDeBrazza'a monkeys were seronegative using a commercially availableHIV-1/2 peptide EIA yet were seroreactive to their homologous IDR and V3MAPs in the assay disclosed herein and were confirmed seropositive usingan HIV-2 WB assay.

Table 3 below shows the sensitivities and specificities of the varioussimian reference groups in terms of reactivities of each specific SIVantibodies against their homologous MAPs. Sensitivities of 100% wereobtained for all but two lineages; specificity was 100% for allpeptides.

TABLE 3 Sensitivities and specificities from non-human reference panelspecimens based on reactivities of each SIV antibodies against theirhomologous MAPs Positive samples Negative samples PEIA + re- PEIA −panel nbr. region Sensitivity nbr. gion specificity SIVsm 16 IDR 16 100%  6+ IDR   6+ 100% 16 V3 16 100% 6 V3 6 100% SIVagm 9 IDR 9 100% 10  IDR10  100% 9 V3 9 100% 10  V3 10  100% SIVsyk 4 IDR 4 100% 2 IDR 2 100% 4V3 3  75% 2 V3 2 100% SIVcpz 3 IDR 3 100% 3 IDR 3 100% 3 V3 2 66.60%   3V3 3 100% SIVl′hoest 2 IDR 2 100% 1 IDR 1 100% 2 V3 2 100% 1 V3 1 100%SIV col 4 IDR 4 100% 31  IDR 31  100% 4 V3 4 100% 31  V3 31  100%

2. Human Reference Panel

There was no significant reactivity observed with the 198 seronegativesamples collected in U.S. blood banks.

The 43 HIV-2 specimens reacted specifically with the SIVsm/HIV-2 gp36MAP as well as the SIVagm MAP. Some cross-reactivities were also foundwith SIVrcm, SIVsyk and to a lesser extend with SIVlhoest. The V3component showed reactivities only to SIVsm and SIVagm peptides.

The specimens from HTLV infected individuals did not show any reactivityto the SIV MAPs, except one of the 44 showed some low reactivity withthe SIVcol peptide with the IDR peptide; no reactivity with V3. Likewiseone of the 18 malaria specimens showed a low reactivity to SIVcpz MAP inIDR while no reactivity was found with the V3. There was no reactivityobserved with any of the 4 specimens obtained from persons infected withmeasles.

With respect to the testing of HIV negative plasma samples fromCameroon, of the samples showing an EIA OD>1 in IDR for an SIV peptide,17.1% were observed in the HE, 7.8% in LE, and 2.3% in G. One samplecollected from our general population reacted to the Colobus peptide inboth IDR (0D=1.250) and V3 (0D=1.798).

In summary, the performance of the presently disclosed MAP assay isefficient in detecting and discriminating primate lentiviral infections.SIV infected simian specimens were correctly identified with the IDRassay, even occasionally exhibiting broad cross-reactivity. Thiscross-reactivity can be a favorable characteristic since it allows oneto detect divergent SIV strains for which sequences were unavailable fordesigning MAPs. The V3 component was more lineage specific inreactivity. Interestingly, the specimen from a person occupationallyinfected with SIVsm (ref) was detected by the assay, indicating that itwill have considerable utility for screening the human population forpotential exposures or infections with SIV strains that hold thepossibility of infecting our blood supply and seed new emerginginfectious diseases. The testing of the HIV negative plasma samples fromCameroon further confirmed that the assay has utility for screeninghuman populations for exposures of infections with SIV strains. Thetesting of the HIV negative samples from Cameroon showed a strongstatistical correlation with exposure to non-human primate blood or bodyfluids or keeping primates as pets.

A total of 93 sera from various species of uninfected monkeys were alsoused for evaluation. All were non reactive to all MAPs, thusdemonstrating the high specificity of the MAP assay. Also specimens fromindividuals with a number of endemic infections did not produce anysignificant reactivity that could compromise the specificity of theassay to SIV only. Concerning HIV positive specimens, cross-reactivitywith SIVcol was remarkably very rare (frequency of only 4.7%), followedby SIVrcm (12.7%) and SIVmnd (14.2%). The highest reactivity was foundwith SIVcpz (90.5%).

The presently disclosed assays proved to be more sensitive than HIVtests and can be used instead of HIV tests for future SIV studies andsentinel surveillance for potential new cross species transmissions ofSIV into humans. The IDR component of the assays is highly sensitive andcan be used in the primary screening of samples. The V3 component ismore specific but less sensitive and can be used if identification tospecies level is sought. The combination of these 2 components thereforeprovides a very effective testing strategy that can be used inserosurveillance, especially for detecting divergent SIV strains inmonkeys as well as SIV-like infections in humans. This is enhanced bythe use of a comprehensive array of peptides covering all geneticallycharacterized primate lentiviruses. The MAP assays offer an open andflexible technology whereby peptides derived from newly identifiednon-human primate SIV strains can be progressively included into thesystem. The possibility of using a wide range of peptides increases theprobability/potential of detecting previously unidentified divergentlentiviral strains. Having illustrated and described the disclosedmethods and assays with reference to several examples, it should beapparent that these methods and assays may be modified in arrangementand detail.

1. An enzyme immunoassay construct, comprising: a first substrate towhich is bound a plurality of detection multiple antigenic peptides,each detection multiple antigenic peptide comprising a portion of animmunodominant region of a transmembrane envelope protein of a primateimmunodeficiency virus, wherein at least one simian immunodeficiencyvirus is represented in at least one of the detection multiple antigenicpeptides; and a second substrate to which is bound a plurality ofdifferentiation multiple antigenic peptides, each differentiationmultiple antigenic peptide comprising a portion of a V3-loop of anenvelope protein of a primate immunodeficiency virus, wherein at leastone simian immunodeficiency virus is represented in at least one of thedifferentiation multiple antigenic peptides; wherein the detectionmultiple antigenic peptide consists of a core matrix and at least twolinear antigenic sequences bonded to the core matrix by β-alanine andd-aspartic acid, each linear antigenic sequence is less than 16 aminoacid residues and the β-alanine and d-aspartic acid serving as spaceramino acids between each linear antigenic sequence and the core matrix;and wherein the differentiation multiple antigenic peptide consists of acore matrix and at least two linear antigenic sequences bonded to thecore matrix by diaminopropionic acid, each linear antigenic sequence isless than 16 amino acid residues and diminopropionic acid serving as aspacer amino acid between each linear antigenic sequence and the corematrix.
 2. The immunoassay of claim 1, wherein the detection multipleantigenic peptide comprises a portion of the immunodominant region ofthe transmembrane protein gp41 or gp36, and the differentiation multipleantigenic peptide comprises a portion of the V3-loop of the envelopeprotein gp120.
 3. The immunoassay of claim 1, wherein each linearantigenic sequence of the detection multiple antigenic peptide comprises5 to 15 amino acid residues, and each linear antigenic sequence of thedifferentiation multiple antigenic peptide comprises 5 to 15 amino acidresidues.
 4. The immunoassay of claim 1, wherein the immunoassay doesnot include any detection multiple antigenic peptide from a humanimmunodeficiency virus and any differentiation multiple antigenicpeptide from a human immunodeficiency virus.
 5. The immunoassay of claim1, wherein the linear antigenic sequence of the detection multipleantigenic peptide is WGCSGKAVCYT (SEQ ID NO: 1).
 6. The immunoassay ofclaim 1, wherein the linear antigenic sequence of the differentiationmultiple antigenic peptide is RGEVQIGPGMTFYNI (SEQ ID NO: 14).
 7. Theimmunoassay of claim 1, wherein the linear antigenic sequence of thedetection multiple antigenic peptide is WGCSGKAVCYT (SEQ ID NO: 1) andthe linear antigenic sequence of the differentiation multiple antigenicpeptide is RGEVQIGPGMTFYNI (SEQ ID NO: 14).
 8. The immunoassay of claim1, wherein the linear antigenic sequence of the detection multipleantigenic peptide consists of a sequence X₁GCX₄X₅X₆X₇X₈CX₁₀T

wherein X₁ is W, I or F; X₄ is S, A or Q; X₅ is G, D, F, W or N; X₆ isK, R, M, S, A; X₇ is A,V or Q; X₈ is V, or I; and X₁₀ is Y, H or R. 9.The immunoassay of claim 1, wherein the detection multiple antigenicpeptide and the differentiation multiple antigenic peptide each consistof four linear antigenic sequences bonded to their respective corematrix.
 10. The immunoassay of claim 1, wherein there are a plurality ofdetection multiple antigenic peptides and a plurality of differentiationmultiple antigenic peptides, and all recognized SIV strain epitopes arerepresented in at least one of the detection multiple antigenic peptideor the differentiation multiple antigenic peptide.
 11. The immunoassayof claim 1, wherein the linear antigenic sequence of the differentiationmultiple antigenic peptide comprises VLPVTIMSGLVFHSQ (SEQ ID NO: 15).12. The immunoassay of claim 1, wherein the linear antigenic sequence ofthe differentiation multiple antigenic peptide comprises VLPVTIMAGLVFHSQ(SEQ ID NO: 16).
 13. The immunoassay of claim 1, wherein the linearantigenic sequence of the differentiation multiple antigenic peptidecomprises IKNIQLAAGYFLPVI (SEQ ID NO: 17).
 14. The immunoassay of claim1, wherein the linear antigenic sequence of the differentiation multipleantigenic peptide comprises EVSTISSTGLLFYYG (SEQ ID NO: 18).
 15. Theimmunoassay of claim 1, wherein the linear antigenic sequence of thedifferentiation multiple antigenic peptide comprises HRNLNTANGAKFYYE(SEQ ID NO: 19).
 16. The immunoassay of claim 1, wherein the linearantigenic sequence of the differentiation multiple antigenic peptidecomprises VKGISLATGVFISLR (SEQ ID NO: 20).
 17. The immunoassay of claim1, wherein the linear antigenic sequence of the differentiation multipleantigenic peptide comprises IVSVPSASGLIFYHG (SEQ ID NO: 21).
 18. Theimmunoassay of claim 1, wherein the linear antigenic sequence of thedifferentiation multiple antigenic peptide comprises YRAVHMATGLSFYTT(SEQ ID NO: 22).
 19. The immunoassay of claim 1, wherein the linearantigenic sequence of the detection multiple antigenic peptide isWGCSGKAVCYT (SEQ ID NO: 1), IGCANMQICRT (SEQ ID NO: 8), or FGCAWRQVCHT(SEQ ID NO: 9).
 20. The immunoassay of claim 1, wherein the linearantigenic sequence of the differentiation multiple antigenic peptideconsists of one of SEQ ID NOS: 14-22.
 21. An enzyme immunoassayconstruct, comprising: a first array of a plurality of detectionmultiple antigenic peptides comprising a portion of an immunodominantregion of a transmembrane protein of a primate immunodeficiency virus;and a second array of a plurality of differentiation multiple antigenicpeptides comprising a portion of a V3-loop of an envelope protein of aprimate immunodeficiency virus; wherein the detection multiple antigenicpeptide consists of a core matrix and at least two linear antigenicsequences bonded to the core matrix by β-alanine and d-aspartic acid,each linear antigenic sequence is less than 16 amino acid residues andthe β-alanine and d-aspartic acid serving as spacer amino acids betweeneach linear antigenic sequence and the core matrix; wherein thedifferentiation multiple antigenic peptide consists of a core matrix andat least two linear antigenic sequences bonded to the core matrix bydiaminopropionic acid, each linear antigenic sequence is less than 16amino acid residues and diminopropionic acid serving as a spacer aminoacid between each linear antigenic sequence and the core matrix and atleast one simian immunodeficiency virus is represented in at least oneof the detection multiple antigenic peptides or the differentiationmultiple antigenic peptides.
 22. The immunoassay of claim 21, whereineach linear antigenic sequence of the detection multiple antigenicpeptide comprises 5 to 15 amino acid residues, and each linear antigenicsequence of the differentiation multiple antigenic peptide comprises 5to 15 amino acid residues.
 23. The immunoassay of claim 21, wherein thelinear antigenic sequence of the detection multiple antigenic peptide isWGCSGKAVCYT (SEQ ID NO: 1) and the linear antigenic sequence of thedifferentiation multiple antigenic peptide is RGEVQIGPGMTFYNI (SEQ IDNO: 14).
 24. The immunoassay of claim 21, wherein the detection multipleantigenic peptide and the differentiation multiple antigenic peptideeach consists of four linear antigenic sequences bonded to theirrespective core matrix.
 25. An enzyme immunoassay construct, comprising:a first substrate to which is bound a plurality of detection multipleantigenic peptides, each detection multiple antigenic peptide comprisinga portion of an immunodominant region of a transmembrane envelopeprotein of a primate immunodeficiency virus, wherein at least one simianimmunodeficiency virus is represented in at least one of the detectionmultiple antigenic peptides; and a second substrate to which is bound aplurality of differentiation multiple antigenic peptides, eachdifferentiation multiple antigenic peptide comprising a portion of aV3-loop of an envelope protein of a primate immunodeficiency virus,wherein at least one simian immunodeficiency virus is represented in atleast one of the differentiation multiple antigenic peptides; whereinthe detection multiple antigenic peptide consists of a core matrix andfour linear detection antigenic sequences bonded to the core matrix byβ-alanine and d-aspartic acid, each linear detection antigenic sequenceis WGCSGKAVCYT (SEQ ID NO: 1) and the β-alanine and d-aspartic acidserve as spacer amino acids between each linear detection antigenicsequence and the core matrix; and wherein the differentiation multipleantigenic peptide consists of a core matrix and four linear antigenicsequences bonded to the core matrix by diaminopropionic acid, eachdifferentiation linear antigenic sequence is RGEVQIGPGMTFYNI (SEQ ID NO:14) and diminopropionic acid serves as a spacer amino acid between eachdifferentiation linear antigenic sequence and the core matrix.